Abstract Enterovirus D68 (EV-D68), a highly contagious non-polio enterovirus, caused the 2014 outbreak affecting thousands of people in multiple countries, including the US, Canada and Europe. Historically, symptoms of EV- D68 infections include rhinorrhea, muscle aches and cough. However, during the 2014 outbreak, there were increasing cases involving more severe respiratory symptoms and hospitalization. Also, because the EV-D68 infections coincided with an increase of acute flaccid myelitis (AFM) cases, EV-D68 is suspected to be associated with AFM even though more data are needed to understand the relationship between them. EV-D68 is currently listed as one of NIAID?s priority pathogens. Unfortunately, there are no FDA-approved drugs or vaccines for the treatments of the EV-D68 infection. Therefore, there is an urgent medical need for more potent therapeutics tailored for EV-D68. The overall goal of this project is to identify and develop small molecule protease inhibitors of EV-D68 2Apro as prophylactics and/or therapeutics for EV-D68 infections and preferably also for infections of the highly related virus EV-A71, which is responsible for hand-foot-and-mouth disease and whose 2Apro shares a conserved sequence and catalytic triad with 2Apro of EV-D68. Our strategy is to address the unmet medical need by identifying small molecule inhibitors targeting the enzymatic activity of a newly characterized EV-D68 protease, 2Apro. The approach is to leverage our experience with 2Apro and homogeneous biochemical screens utilizing FRET to identify small molecules that inhibit the enzymatic activity of 2Apro. In Preliminary Studies, we developed a FRET assay for EV-D68 2Apro and have applied the assay in a low/medium-throughput screen of ~2000 compounds with Z?-factors ?0.7. The pilot screen identified telaprevir as an inhibitor of EV-D68 2Apro with confirmed antiviral activity against a panel of contemporary EV-D68 strains. In Phase I, for Aim 1, an FRET HTS will be optimized and applied to diverse chemical libraries for the identification of small molecules that inhibit the enzymatic activity of 2Apro. In Aim 2, FRET secondary assays, a thermal shift assay and a cell-based CPE assay will be optimized and used to validate confirmed hits, to determine the binding affinity, and to measure in vitro antiviral activity against strains of serotypes D68 and A71. In Aim 3, validated hits prioritized based on their drug- likeness and antiviral spectrum will be evaluated for their ADME properties. We will also explore preliminary SAR of prioritized validated hits. In Phase II, we will chemically optimize priority inhibitors for potency and selectivity, in vitro and in vivo pharmacokinetic properties, and evaluate them in animal infection models.